Our specific aim is to study the enzymology of protein kinases that recognize and phosphorylate tyrosine residues. Increases in tyrosine phosphorylation have heretofore been found associated with the increased rates of cellular proliferation that accompany the malignant transformation of cells by certain oncogenic RNA tumor viruses and the action of some mammalian growth factors. The raison d'etre for this association is unclear. We feel that a useful model system is needed for studying tyrosine protein kinases that are under normal cellular control. The system we have chosen is that of lymphocyte activation, which provides us with a population of cells whose normal response to external stimuli is to proliferate and differentiate. This system also allows the comparison of transformed cells, lymphomas, and leukemias with their normal but proliferating counterparts. Our preliminary results suggest that lymphocytes have higher levels of membrane-associated tyrosine protein kinase activity than do other murine cell types. We plan to isolate lymphocyte subpopulations, stimulate them to proliferate in vitro with polyclonal mitogens and lymphocyte growth factors and determine the effects of these agents on tyrosine protein kinase activity and the phosphorylation of membrane-associated substrates. The molecular basis for any observed changes will be investigated, and the kinases involved isolated and characterized. We intend to investigate the cellular distribution, subcellular localization, and mechanisms of regulation of these tyrosine protein kinases. The methodologies to be employed include: (1)\the isolation and fractionation of lymphocyte populations; (2)\in vitro cell culture and mitogen and growth factor activation; (3)\ATP:protein phosphotransferase assays; (4)\enzyme isolation and characterization; and (5)\photoaffinity labeling. We hope that through a greater understanding of the biochemistry of tyrosine protein kinases from normal lymphocytes we can begin to define the pathways involved in the regulation of cellular proliferation in order to identify the lesions that result within this pathway that can lead to malignant growth. (LB)